Endogenous nitrosation of amino acids and related molecules is an important potential source of carcinogenic N-nitroso compounds in human populations. N-nitrosoproline (NPro), a noncarcingenic, nonmutagenic substance has been established as a stable index for endogenous nitrosation. Ingested, preformed NPro is excreted unchanged in human and animal urine, and the concentration of NPro inhuman and animal urine has been shown to increase proportionally with increasing amounts of precursor nitrates and L-proline consumed, respectively, in food stuffs and drinking water. Moreover, preliminary studies in humans indicate that higher level of NPro may be found in smokers as compared to nonsmokers. Similar results are found in individuals from geographical areas of high risk for stomach and esophageal cancer compared to those from areas of low risk for these diseases. Current knowledge and recent information of endogenous nitrosation was obtained using gas chromatography and terminal energy analyzer measurements of NPro in urine. This method detects a minimum of 0.2 ng NPro per ml which is insufficient for detection of serum levels of NPro. In this project, we intend to develop an enzyme immunoassay (EIA) and an ultrasensitive enzymatic radio-immunoassay (USERIA) with femtomole sensitivity and the possibility to use both urine and sera to measure NPro in vivo in human populations of interest to the study of human carcinogenesis.